Cell adhesion is critical to the viability of living organisms. Adhesion holds multicellular tissues together and directs embryonic development. It plays important roles in wound healing, eradication of infection and blood coagulation. Integrins are a family of cell surface proteins intimately involved in all of these functions. They have been found in nearly every type of human cell except red blood cells. Abnormalities in integrin function contribute to a variety of disorders including inflammatory diseases, heart attack, stroke, and cancer.
Integrins consist of heterodimers of α and β subunits, non-covalently bound to each other. These cell surface receptors extend through the cell membrane into the cytoplasm. At least 15 different α and 9 different β subunits are known. However, because most α proteins associate with only a single β there are about 21 known integrin receptors. On the cell surface the heads of the two subunits contact each other to form a binding surface for extracellular protein ligands, allowing attachment to other cells or to the extracellular matrix. The affinity of these receptors may be regulated by signals from outside or within the cell. For example, recruitment of leukocytes to the site of injury or infection involves a series of adhesive interactions. Weak interaction between endothelial and leukocyte selectins and carbohydrates mediate transient adhesion and rolling of the leukocyte along the vessel wall. Various chemokines and other trigger factors released by the site of inflammation serve as signals to activate integrins from a quiescent to a high affinity state. These activated integrins then bind their cognate ligands on the surface of the endothelial cells, resulting in strong adhesion and flattening of the leukocyte. Subsequently the leukocyte migrates through the endothelium into the tissue below.
Integrin α4β1 mediates cell adhesion primarily through binding to either vascular cell adhesion molecule-1 (VCAM-1) or an alternatively spliced variant of fibronectin containing the type III connecting segment (IIICS). A variety of cells involved in inflammation express α4β1, including lymphocytes, monocytes, basophils and eosinophils, but not neutrophils. Monoclonal antibodies to the α4 subunit have been used to validate α4-containing integrins as potential therapeutic targets in animal models of rheumatoid arthritis (Barbadillo et al. Springer Semin Immunopathol 16: 427-36, 1995; Issekutz et al. Immunology 88: 569-76, 1996), acute colitis (Podolsky et al. J Clin Invest 92: 372-80, 1993), multiple sclerosis (Yednock et al. Nature 356: 63-6, 1992), asthma (Abraham et al. J. Clin. Invest. 93: 776-87, 1994; U.S. Pat. No. 5,871,734) and diabetes (Tsukamoto et al. Cell Immunol 165: 193-201, 1995). More recently, low molecular weight peptidyl derivatives have been produced as competitive inhibitors of α4β1 and one has been shown to inhibit allergic airway responses in sheep (Lin et al. J Med Chem 42: 920-34, 1999).
It has been shown that a key sequence in MCS involved in binding to α4β1 is the 25 residue peptide CS1, and within that sequence the minimally recognized motif is the tripeptide, LDV. A similar sequence, IDS, has been implicated in the binding of VCAM-1 to α4β1. X-ray crystal structures of an N-terminal two-domain fragment of VCAM-1 show that the IDS sequence is part of an exposed loop linking two beta-strands (Jones et al. Nature 373: 539-44, 1995; Wang et al. Proc Natl Acad Sci USA 92: 5714-8, 1995). Cyclic peptides and derivatives thereof which adopt reverse-turn conformations have proven to be inhibitors of VCAM-1 binding to α4β1 (WO 96/00581; WO 96/06108; Doyle et al. Int J Pept Protein Res 47: 427-36, 1996). In addition, a number of potent and selective (versus α5β1) cyclic peptide-based inhibitors have been discovered (Jackson et al. J Med Chem 40: 3359-68, 1997). Several non-peptidyl beta-turn mimetics have also been reported to bind α5β1 with IC50s in the low micromolar range (Souers et al. Bioorg Med Chem Lett 8: 2297-302, 1998). Numerous phenylalanine and tyrosine derivatives have also been disclosed as inhibitors of α4β1 (WO 99/06390; WO 99/06431; WO 99/06433; WO 99/06434; WO 99/06435; WO 99/06436; WO 99/06437; WO 98/54207; WO 99/10312; WO 99/10313; WO 98/53814; WO 98/53817; WO 98/58902) However, no potent and orally available small molecule inhibitors have been disclosed.
A related integrin, α4β7, is expressed on the surface of lymphocytes and binds VCAM-1, fibronectin and mucosal addressin cell adhesion molecule 1 (MAdCAM-1). Integrin α4β7 and MAdCAM mediate recirculation of a subset of lymphocytes between the blood, gut, and lymphoid tissue. Similar to VCAM-1 and Fibronectin CS-1 there is a tripeptide sequence, LDT, present on the CD loop of MAdCAM-1 which is important for recognition by α4β7. An X-ray crystal structure shows this sequence is also part of a turn structure (Tan et al. Structure 6: 793-801, 1998). Recent studies have shown that α4β7 may also play a part in diseases such as asthma (Lobb et al. Ann N Y Acad Sci 796: 113-23, 1996), inflammatory bowel disease (Fong et al. Immunol Res 16: 299-311, 1997), and diabetes (Yang et al. Diabetes 46: 1542-7, 1997). In addition, while α4 integrins appear to be down-regulated in carcinomas such as cervical and prostate, they appear to be up-regulated in metastatic melanoma (Sanders et al. Cancer Invest 16: 329-44, 1998), suggesting that inhibitors of α4β1 and α4β7 may be useful as anticancer agents.
Reverse-turns comprise one of three classes of protein secondary structure and display three (gamma-turn), four (beta-turns), or more (loops) amino acid side chains in a fixed spatial relationship to each other. Turns have proven important in molecular recognition events (Rose et al. Advances in Protein Chemistry 37: 1-109, 1985) and have engendered a burgeoning field of research into small molecule mimetics of them (e.g. Hanessian et al. Tetrahedron 53: 12789-12854, 1997). Many mimetics have either been external turn-mimetics which do not allow for the display of all the physiologically relevant side-chains (e.g. Freidinger et al. Science 210: 656-8, 1980) or small, conformationally mobile cyclic peptide derivatives (e.g. Viles et al. Eur J Biochem 242: 352-62, 1996). However, non-peptide compounds have been developed which closely mimic the secondary structure of reverse-turns found in biologically active proteins or peptides. For example, U.S. Pat. Nos. 5,475,085, 5,670,155 and 5,672,681, to Kahn and published PCT WO94/03494 to Kahn all disclose conformationally constrained, non-peptidic compounds which mimic the three-dimensional structure of reverse-turns. More recently, published PCT WO97/15577 to Kahn and PCT WO98/49168 to Kahn et al. have disclosed additional, highly constrained bicyclic heterocycles as reverse-turn mimetics. Nevertheless, as no one template can mimic every type of turn, there remains a need in the art for additional reverse-turn templates and methods for their use.
While significant advances have been made in the synthesis and identification of conformationally constrained, reverse-turn mimetics, there is still a need in the art for small molecules that mimic the secondary structure of peptides. In addition, there is a need in the art for techniques for synthesizing libraries of such mimetics and screening the library members against biological targets to identify bioactive library members. Further, there is a need in the art for small, orally available inhibitors of integrins, for use in treating inflammatory diseases and cardiovascular diseases, as well as some cancers. In particular there is a need for inhibitors of α4β1 and α4β7, for use in the treatment of rheumatoid arthritis, asthma, diabetes and inflammatory bowel disease. The present invention fulfills these needs, and provides further related advantages.